After the first week decline, HIV in the four patients with higher baseline HIV RNA (3.4–4.5 log10 copies/ml) reached a minimum level between the second and third week (Table 2). A rebound in HIV from that time to the end of treatment wass observed in these four patients (Fig. 1), although in two of them HIV viral load at the end of treatment (48 weeks) was 1 log10 lower than baseline. Conversely, in the four patients with lower baseline HIV load (< 3.3 log10), HIV viral load fell below the level of detection (< 50 copies/ml) by days 3–42 and stayed at that level until the end of treatment. At the end of treatment, HIV levels rebounded back to baseline for all patients, reiterating that the consistent viral suppression is a direct effect of the PEG–IFN and ribavirin therapy.
The significant qualitative differences between HIV and HCV kinetics in the same patients indicate that PEG–IFN possibly has a different antiviral mechanism for these two viruses. HCV kinetics seen in these nine patients are consistent with the assumption that IFN blocks HCV production from infected cells  as a function of PEG–IFN pharmacokinetics [25,26]. In contrast, HIV kinetics are not readily explained by fitting the data with the assumption that the major antiviral effect of IFN is blocking virion production. Simulation of a mathematical model of viral dynamics (Fig. 3a, Eqs 1–4) shows that blocking virion production gives rise to a bi-phasic decline (Fig. 3b), where the first phase slope is governed by the half life of free virus and IFN dose or concentration  and the second phase slope is determined by the half life of infected cells (scaled by the effectiveness in blocking production). Such bi-phasic decline is observed here for HCV but not for HIV.
Nevertheless, it is possible to fit the HIV decline assuming a large blocking virion production effect (ε = 0.76–0.97) and thus making the decline during the whole first week part of the first phase (Fig. 3c, d). However, to fit the data in that case, HIV free virus half life must be very long (shortest estimate of 10.4–20.8 h, maximal estimate of c = 0.8–1.6, Table 2), which is significantly longer than the half life of 2–6 h known from previous studies during ART  or 0.5–2 h found during apheresis . Alternatively, it is also possible to fit HIV decline by assuming a low effectiveness in blocking virion production (ε = 0.22–0.58), without blocking de novo infection (η = 0.0), thus obtaining a very small first phase decline (Fig. 3c,d). However, in that case the half life of infected cells needs to be in the range of 0.3–0.8 days (minimal estimate of δ = 0.8–2.2, Table 2), in order to fit HIV decline during the first week, which is again inconsistent with the half life of 1.1–1.8 days shown in previous studies [22,27,29]. Thus, we conclude that blocking virion production/release from infected cells as the major antiviral effect of IFN against HIV cannot explain the kinetics of HIV during PEG–IFN/ribavirin treatment.
On the other hand, if one assumes that the major (or even the only) effect of IFN on HIV is blocking de novo infection then it is possible to fit the data for HIV decline with half-lives of free virus and of infected cells that are consistent with the values described in the literature (Fig. 3e,f, Table 2). Due to the infrequent sampling during the first day it is not possible to obtain an accurate estimate of the free virus half life (or of c), but any value between 2 and 8 h allows a good fit. Also, similar to previous studies , since the parameters η and δ are coupled (Eq. 4), only their product, and the half life of the decline slope, can be estimated. However, an effectiveness of blocking infection in the range 70–100% gives a reasonable estimate of the half life of productively infected cells. The non-linear best fit of the data when allowing blocking both de novo infection and virion production (Fig. 3e,f, Table 2) shows that a low effectiveness of blocking virion production (mean ε = 31%) is possible together with a larger effectiveness of blocking de novo infection.
The decline in HIV RNA was independent of IFN concentration. Also, in eight of nine patients HIV RNA decline did not show a pharmacokinetic effect due to the decline in IFN levels at days 1–7 after the injection (three patients have HIV RNA<50 cp/ml from day 3 or 7 on; four patients had slightly slower decline at days 5–7 compare to days 1–5 but the slowing down continued also at days 7–10 when IFN levels increased again; one patient had little decline during the week). Rather, high baseline HIV RNA was highly associated with a rebound in HIV RNA that started at day 10 or 14. Nevertheless, a significant correlation (r, 0.9; P < 0.002) was found between HIV decline at days 3–7 and the half life of IFN (Fig. 2c), indicating that pharmacokinetic effects may also play a partial role in determining the rebound. Indeed, the one patient (I1) with apparent pharmacokinetic related viral rebound at days 3–7 and 10–14 had the shortest IFN half life (1.3 days) in the cohort.
The above results indicate a high sensitivity of HIV to the IFN effect in blocking de novo infection, such that the 90% effective IFN concentration is low enough (maximal estimate of ηc90 of the order of 1000–3000 pg/ml) for the decline in IFN levels over time to not play a significant role. Unfortunately, due to the non-frequent sampling it is difficult to obtain an accurate estimate of ηc90 in all patients. Nevertheless, we were able to fit HIV kinetics as a function of the IFN pharmacokinetics in one patient (Fig. 3g), in parallel to fitting HCV kinetics as a function of the same pharmacokinetic profile (Fig. 3h). As above, the results show that if one assumes that HIV is more sensitive to blocking virion production by IFN than blocking infection then the half-lives of free virions or of the infected cells are not in agreement with previously described data. On the other hand, if one allows for HIV to be more sensitive to blocking infection by HIV (ηc90, 5400 pg/ml and Nη, 5.7 versus εc90, 93 000 pg/ml and Nε, 1.7) then a fit of the viral rebound as function of IFN pharmacokinetics (IFN0, 36 400; ka, 1.54; ke, 0.54) is obtained with appropriate half-lives (2–6 h for free virus and 1.1 days for infected cells). Interestingly, HCV was more sensitive to blocking production by IFN (εc90, 9900 pg/ml and Nε, 2), but unfortunately, as previously shown , it is not possible to estimate the effectiveness in blocking infection for HCV. Note that these parameter values are to be used only qualitatively as a range of values allows the data to fit due to the non-frequent sampling and the large number of parameters involved.
To test the above hypothesis we performed HIV replication suppression experiments using PEG–IFN in vitro. When U87 cells were used as targets for luciferase-pseudotype R5 and X4 viruses, PEG–IFN treatment resulted in a dose-dependent suppression of HIV replication of both R5 and X4 strains (Fig. 4a,b). Incubation with PEG–IFN prior to infection provided superior suppression of HIV replication when compared to treatment of target cells after infection. When low doses of PEG–IFN were added after infection, the suppression was minimal or not observed, whereas when the same PEG–IFN doses were used preincubation suppression of HIV replication was 40–80%.
Similar results were obtained when real-time PCR was used to detect HIV copy numbers after infection of CD8 depleted PBMC with R5 virus (Fig. 4c). Again, this dichotomous suppressive effect was more pronounced with preincubation of target cells with IFN than with IFN treatment postinfection, which is consistent with our in vivo results that the predominant anti-HIV effect of IFN is to block de novo infection rather than block production from already infected cells.
HIV and HCV exhibit significantly different viral kinetics during treatment with PEG–IFN-α-2b, possibly indicating that IFN has a different antiviral effect against HIV than it does against HCV. Mathematical modeling of our data indicates that the major effect of IFN against HIV is to block de novo infection, while the major IFN effect against HCV is blocking virion production. This conclusion is corroborated by in vitro studies showing that IFN induces more potent suppression of HIV replication when it is preincubated prior to infection of target cells with HIV, rather than IFN treatment postinfection with HIV. Thus, IFN seems to have a predominant preintegration effect rather than postintegration in suppressing HIV replication in vitro, consistent with our in vivo results.
Indeed, a recent study suggested that the major antiviral effect of IFN is mediated by induction of APOBEC3G genes in monocytes  and in resting primary T lymphocytes . APOBEC3G is an innate host intracellular antiviral factor that is constitutively expressed on resting T cells and monocytes [31,33,34]. APOBEC3G functions as a cytidine deaminase, which catalyzes the deamination of cytosine to uracil, leading to accumulation of adenine nucleotides into the viral DNA and resulting in incorporation of lethal mutations into the HIV genome [35–38]. These results suggest that IFN induces up-regulation of an innate anti-HIV mechanism at the level of preintegration. Indeed, it has been shown that IFN-α induces APOBEC3A in lymphocytes, thereby suppressing HIV replication (Peng et al. unpublished data). The exact mechanism by which APOBEC3A suppresses HIV replication is not yet clearly understood, but our results would suggest that it also acts at the preintegration level. Induction of host innate antiviral machinery is a novel approach to develop newer therapeutic agents, and our study indicates that such mechanisms can indeed be a viable option in the treatment of HIV infection.
When the antiviral effect of IFN is compared to that described with the use of protease inhibitor (PI)-containing ART, the magnitude and durability of HIV viral load suppression is quite similar [27,29]. However, the IFN-induced antiviral effect seem to be inversely related to the baseline viral levels, while there is a direct relationship between the baseline HIV viremia and antiviral response to PI-containing ART [27,29]. These findings suggest that IFN may have a completely different antiviral mechanism against HIV than that described with ART. The lack of HIV decline in patient I5, who had the highest baseline HIV viral load, and the rebound found in patients with higher baseline viral load indicate that the IFN-induced antiviral effect is higher in patients with relatively low levels of HIV replication. It should be further verified if preexisting high levels of endogenous IFN in patients with high viral load potentially gives rise to insensitivity to IFN treatment and may explain the lack of response to IFN in these patients . These findings also suggest that IFN may have a complementary antiviral mechanism against HIV to that described with ART, and that their combination may have a synergistic effect in suppressing HIV replication in vivo. Such therapeutic approaches will expand the options available to clinicians to target HIV and attain maximal suppression of HIV replication.
IFN has been used as an anti-HIV agent in the past and in some recent studies as well [17,18] However, most clinicians have reservations about using IFN as an anti-HIV agent due to its adverse event profile . IFN use is associated with several serious adverse events which are much more pronounced in HIV-infected individuals . However, our study implies that IFN does have significant anti-HIV activity and could have a role in the treatment of HIV infection. Recent advances in developing newer IFN formulations (for example, albumin conjugated IFN [41,42]) could enable clinicians to administer IFN every 2–4 weeks with fewer adverse events. Furthermore, our pharmacodynamical results indicate that although low levels of IFN may suffice to give rise to an HIV decline, nevertheless an improved pharmacokinetic profile  could improve the antiviral effect of IFN. A significant number of patients develops treatment failure in response to ART, due to the emergence of antiviral drug resistance, and exhibit consistent low levels of viremia . An IFN product could suppress HIV replication in these patients in combination with other antiretroviral agents and prevent the development of complete viral resistance. Clinical trials to test this hypothesis will be valuable in expanding our therapeutic options against HIV infection.
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